The coagulation factors VII and X circulate in a precursor or zymogen form in the blood. Although readily available for participation in coagulation, the sequence must first be initiated by the activation of tissue factor (TF), a membrane-bound glycoprotein found on the plasma membranes of a variety of cells including circulating monocytes. The exact nature of monocyte TF (MTF) activation remains largely unknown. Monocyte procoagulant activity can be dramatically enhanced by stimulation with immune complexes, a fragment of C 5, a T cell-derived mediator, or bacterial endotoxin. Since TF is readily available to the circulating coagulation proteins, yet fails to induce rampant coagulation, there must be a mechanism for the regulation of its presentation to the coagulation zymogens. We will examine the mechanism(s) of tissue factor activation in the following sequence. (1) Partially purified human brain TF will be used to generate monoclonal antibodies (mAbs) to human TF. (2) Monoclonal Abs will be used to facilitate TF purification by affinity chromatography and the pure TF will be used for the development of a direct solid phase assay for subsequent mAbs. (3) Monoclonal Abs will then be used to characterize the molecular mechanism(s) of TF activation. Two models will be examined--a quantitative increase in TF molecules on the cell surface and a qualitative change in TF structure by proteolysis or conformational change. Quantitative amplification of TF on cell membranes will be assessed with quantitative immunologic assays. TF structural changes will be assessed with two-dimensional electrophoresis following differential radiolabeling. (4) Unique mAb will be sought in inhibition assays designed to detect mAb to functionally specific sites on the TF molecule, including the binding sites for factors VII, X, calcium, and Concanavalin A. These unique mAbs will be used to examine the ordering of the TF-VII-X complex formation, the role of calcium in complex formation, and the exposure of the TF glycoprotein during cell activation.